Study On Cloning And Expression Of The Fusion Gene Cry2Aa-gna

Bacillus thuringiensis(Bt) is a very important entomopathogen.It can produce specifically toxic crystal proteins able to kill many agricultural pests,including lepidopteran,dipteran,coleopteran,hymenopteran,acarian and nematodes.Since 1980s,Bt genes expressing toxic proteins were inserted to plant by recombinant DNA technology,and got insect resistant Bt crops.Bt crops has not only reaped significant economic benefits,but also resulted in good social and ecological benefits.However,there were some shortcomings for Bt crops,such as narrow target insect spectrum,low toxicity and tolerance evolution of some insects.To solve these problems,this study aimed to fuse Bacillus thuringiensis gene and Galanthus nivalis agglutinin(GNA) gene as to include piercing-sucking pests onto the target insect range of insecticidal crop inserted with fused gene.Using Polymerase Chain Reaction and TA cloning techniques,the cry2Aa gene and gna gene was cloned into pGEM-T Easy vector and named pGEM-T-C1 and pGEM-T-G1 separately.The length of cry2Aa gene and gna gene were 1899bp and 342bp separaterly. Through double digest and ligation reaction,pET-C1 vector,pET22b-G1 vector and pET-C-G vector have been constructed and expressed in Escherichia coli Rosetta(DE3) strain by the use of pET22-b(+) vector prokaryotic expression system.The results of SDS-PAGE and Western-Blot confirmed that these three expressive plasmids which transformed Escherichia coli can express Cry2Aa proteins(63kDa),GNA proteins(12kDa) and Cry2Aa-GNA fusion proteins(75kDa) highly.